MultiGreen: A multiplexing architecture for GreenGate cloning.

Genetic modification of plants fundamentally relies upon customized vector designs. The ever-increasing complexity of transgenic constructs has led to increased adoption of modular cloning systems for their ease of use, cost effectiveness, and rapid prototyping. GreenGate is a modular cloning system catered specifically to designing bespoke, single transcriptional unit vectors for plant transformation-which is also its greatest flaw.

A source of resistance against yellow mosaic disease in soybeans correlates with a novel mutation in a resistance gene.

Yellow mosaic disease (YMD) is one of the major devastating constraints to soybean production in Pakistan. In the present study, we report the identification of resistant soybean germplasm and a novel mutation linked with disease susceptibility. Diverse soybean germplasm were screened to identify YMD-resistant lines under natural field conditions during 2016-2020.

Editing efficiencies with Cas9 orthologs, Cas12a endonucleases, and temperature in rice.

The advent of CRISPR-Cas technology has made it the genome editing tool of choice in all kingdoms of life, including plants, which can have large, highly duplicated genomes. As a result, finding adequate target sequences that meet the specificities of a given Cas nuclease on any gene of interest remains challenging in many cases. To assess target site flexibility, we tested five different Cas9/Cas12a endonucleases (SpCas9, SaCas9, St1Cas9, Mb3Cas12a, and AsCas12a) in embryogenic rice calli from Taipei 309 at 37°C (optimal temperature for most Cas9/Cas12a proteins) and 27°C (optimal temperature for tissue culture) and measured their editing rates under regular tissue culture conditions using Illumina sequencing.

Transgenic Soybeans Expressing Phosphatidylinositol-3-Phosphate-Binding Proteins Show Enhanced Resistance Against the Oomycete Pathogen .

Oomycete and fungal pathogens cause billions of dollars of damage to crops worldwide annually. Therefore, there remains a need for broad-spectrum resistance genes, especially ones that target pathogens but do not interfere with colonization by beneficial microbes. Motivated by evidence suggesting that phosphatidylinositol-3-phosphate (PI3P) may be involved in the delivery of some oomycete and fungal virulence effector proteins, we created stable transgenic soybean plants that express and secrete two different PI3P-binding proteins, GmPH1 and VAM7, in an effort to interfere with effector delivery and confer resistance.

Two efficient CRISPR/Cas9 systems for gene editing in soybean.

Genome editing using CRISPR/Cas9 has been highlighted as a powerful tool for crop improvement. Nevertheless, its efficiency can be improved, especially for crops with a complex genome, such as soybean. In this work, using the CRISPR/Cas9 technology we evaluated two CRISPR systems, a one-component vs.

Effects of type I Diacylglycerol O-acyltransferase (DGAT1) genes on soybean (Glycine max L.) seed composition.

Type I Diacylglycerol acyltransferase (DGAT1) catalyzes the final step of the biosynthesis process of triacylglycerol (TAG), the major storage lipids in plant seeds, through the esterification of diacylglycerol (DAG). To characterize the function of DGAT1 genes on the accumulation of oil and other seed composition traits in soybean, transgenic lines were generated via trans-acting siRNA technology, in which three DGAT1 genes (Glyma.13G106100, Glyma.

Identification of functional single nucleotide polymorphism of and determination of its transcriptional effect.

In plants, the phenylpropanoid pathway is responsible for the synthesis of a diverse array of secondary metabolites that include lignin monomers, flavonoids, and coumarins, many of which are essential for plant structure, biomass recalcitrance, stress defense, and nutritional quality. Our previous studies have demonstrated that PtrEPSP-TF, an isoform of 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, has transcriptional activity and regulates phenylpropanoid biosynthesis in . In this study, we report the identification of single nucleotide polymorphism (SNP) of that defines its functionality.

Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis.

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants.

Systematic Mutagenesis of Serine Hydroxymethyltransferase Reveals an Essential Role in Nematode Resistance.

is a major genetic locus that contributes to soybean cyst nematode (SCN) resistance in the Peking-type resistance of soybean (), which also requires the gene. By map-based cloning and functional genomic approaches, we previously showed that the gene encodes a predicted cytosolic serine hydroxymethyltransferase (GmSHMT08); however, the novel gain of function of in SCN resistance remains to be characterized. Using a forward genetic screen, we identified an allelic series of mutants that shed new light on the mechanistic aspects of -mediated resistance.

Ketocarotenoid Production in Soybean Seeds through Metabolic Engineering.

The pink or red ketocarotenoids, canthaxanthin and astaxanthin, are used as feed additives in the poultry and aquaculture industries as a source of egg yolk and flesh pigmentation, as farmed animals do not have access to the carotenoid sources of their wild counterparts. Because soybean is already an important component in animal feed, production of these carotenoids in soybean could be a cost-effective means of delivery. In order to characterize the ability of soybean seed to produce carotenoids, soybean cv.

Targeted genome modifications in soybean with CRISPR/Cas9.

Background: The ability to selectively alter genomic DNA sequences in vivo is a powerful tool for basic and applied research. The CRISPR/Cas9 system precisely mutates DNA sequences in a number of organisms. Here, the CRISPR/Cas9 system is shown to be effective in soybean by knocking-out a green fluorescent protein (GFP) transgene and modifying nine endogenous loci.

Simple gene silencing using the trans-acting siRNA pathway.

In plants, particular micro-RNAs (miRNAs) induce the production of a class of small interfering RNAs (siRNA) called trans-acting siRNA (ta-siRNA) that lead to gene silencing. A single miRNA target is sufficient for the production of ta-siRNAs, which target can be incorporated into a vector to induce the production of siRNAs, and ultimately gene silencing. The term miRNA-induced gene silencing (MIGS) has been used to describe such vector systems in Arabidopsis.

Biolistic transformation of elite genotypes of switchgrass (Panicum virgatum L.).

Transformation of elite switchgrass (Panicum virgatum L.) genotypes would facilitate the characterization of genes related to cell wall recalcitrance to saccharification. However, transformation of explants from switchgrass plants has remained difficult.

Gateway-compatible vectors for high-throughput gene functional analysis in switchgrass (Panicum virgatum L.) and other monocot species.

Switchgrass (Panicum virgatum L.) is a C4 perennial grass and has been identified as a potential bioenergy crop for cellulosic ethanol because of its rapid growth rate, nutrient use efficiency and widespread distribution throughout North America. The improvement of bioenergy feedstocks is needed to make cellulosic ethanol economically feasible, and genetic engineering of switchgrass is a promising approach towards this goal.

The rice miniature inverted repeat transposable element mPing is an effective insertional mutagen in soybean.

Insertional mutagenesis of legume genomes such as soybean (Glycine max) should aid in identifying genes responsible for key traits such as nitrogen fixation and seed quality. The relatively low throughput of soybean transformation necessitates the use of a transposon-tagging strategy where a single transformation event will produce many mutations over a number of generations. However, existing transposon-tagging tools being used in legumes are of limited utility because of restricted transposition (Ac/Ds: soybean) or the requirement for tissue culture activation (Tnt1: Medicago truncatula).

Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation.

Background: The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (Panicum virgatum L.) ubiquitin genes (PvUbi1 and PvUbi2) were cloned and characterized.

Bacterial citrate synthase expression and soil aluminum tolerance in transgenic alfalfa.

Alfalfa is very sensitive to soil acidity and its yield and stand duration are compromised due to inhibited root growth and reduced nitrogen fixation caused by Al toxicity. Soil improvement by liming is expensive and only partially effective, and conventional plant breeding for Al tolerance has had limited success. Because tobacco and papaya plants overexpressing Pseudomonas aeruginosa citrate synthase (CS) have been reported to exhibit enhanced tolerance to Al, alfalfa was engineered by introducing the CS gene controlled by the Arabidopsis Act2 constitutive promoter or the tobacco RB7 root-specific promoter.